Cell surface serine proteases as regulators of epithelial development, repair, and malignancy Background: Cell behavior in is regulated by proteases and protease inhibitors that operate in the pericellular environment to provide cytokine/growth factor maturation, matrix remodeling, signaling receptor activation ion channel activity, and more. We aim to to understand the molecular functions of membrane-anchored serine proteases and their inhibitors in vertebrate development, epithelial homeostasis, and epithelial carcinogenesis. Differential regulation of matriptase-prostasin cascade by HAI-1 and HAI-2 Previous research by us and by others has shown that the membrane-anchored serine proteases, matriptase and prostasin, are ubiquitously co-expressed in vertebrate epithelia, and that they constitute a proteolytic cascade system that is critical for the development and homeostasis of multiple epithelia, including oral squamous epithelium. We have also shown that the closely related Kunitz-type transmembrane serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are essential regulators of the matriptase-prostasin cascade. HAI-1 and HAI-2 are near-ubiquitously co-expressed in epithelial cells, are expressed in high molar excess over matriptase, and display identical inhibitor profiles in vitro, all in all making the strict requirement for expression of both inhibitors in the same cell for epithelial integrity somewhat of a conundrum. We defined the differential functions of HAI-1 and HAI-2 in regulating the matriptase-prostasin cascade by genetically downregulating the two inhibitors in intact mouse epithelia of the small and large intestine and in cultured epithelial monolayers and determine the effect of individual deficiency of HAI-1 and HAI-2 on the trafficking and activation of matriptase and prostasin. Loss of endogenous HAI-1 did not affect matriptase levels or subcellular localization in epithelial cells of intact intestinal tissues. In contrast, the level of endogenous intestinal epithelial matriptase protein was markedly decreased by the absence of HAI-2 in the absence of changes in the matriptase mRNA level, suggesting HAI-2 regulates endogenous matriptase expression at the translational or post-translational level. To understand this phenomenon at the molecular level, we silenced HAI-1 and HAI-2 in intestinal cell monolayers. As we observed in intact intestines, silencing of HAI-1 did not affect matriptase expression or location, while silencing of HAI-2 caused a dramatic decrease in cell-associated matriptase and a accumulation of activated matriptase in the conditioned medium in complex with HAI-1. Simultaneous silencing of prostasin prevented the increased activation and shedding of matriptase caused by HAI-2 silencing. These findings suggest that HAI-2 primarily serves to prevent premature activation of the matriptase-prostasin cascade within the secretory pathway, while HAI-1 serves to regulate the activity of cell surface matriptase and, possibly, prostasin. A role of matriptase in established squamous cell carcinoma High matriptase expression has been correlated with poor disease outcome in oral squamous cell carcinoma. Furthermore, we have previously shown that mis-expression of matriptase in squamous epithelium causes malignant transformation. This oncogenic property of matriptase is linked to the dual proteolytic activation of pro-hepatocyte growth factor-cMet-Akt-mTor proliferation/survival signaling and PAR-2-G&#945;i-NF&#954;B inflammatory signaling. In these prior studies, matriptase was congenitally and constitutively deregulated in mice. To examine if matriptase constitutes a potential therapeutic target, we generated triple-transgenic mice with constitutive deregulation of matriptase and simultaneous inducible expression of the potent cognate matriptase inhibitor, HAI-2. Interestingly, the induction of HAI-2 in established tumors markedly impaired malignant progression and caused regression of individual tumors. Tumor regression correlated with reduced accumulation of inflammatory cells, likely caused by diminished expression of pro-tumorigenic inflammatory cytokines. The data suggest that matriptase-mediated cell signaling could be a therapeutic target for both squamous cell carcinoma chemoprevention and for the treatment of established tumors. Reengineered bacterial cytotoxins as antitumor and protease imaging agents Background: Elevated expression of matrix degrading proteases is a hallmark of human cancer. We work on the development of reengineered bacterial cytotoxins, activated by proteases expressed in the tumor microenvironment, as novel therapeutic agents for cancer and as tools for the imaging of specific cell surface proteolytic activity. Development of an intercomplementing toxin with improved therapeutic index Anthrax lethal toxin (LT) consists of the cellular binding moiety, protective antigen (PrAg), and the catalytic moiety, lethal factor (LF). To target cells, PrAg binds to the ubiquitously expressed cell-surface receptors, TEM8 and CMG2, and is then proteolytically processed by furin to form a LF-binding competent PrAg oligomer where each LF binding site is comprised of three subsites on two adjacent PrAg monomers. By exploiting the requirement of LF for binding to two adjacent PrAg molecules, we previously generated an intercomplementing PrAg variant requiring dual activation by co-localized urokinase plasminogen activator (uPA) and matrix metalloproteinase (MMP) activity to intoxicate target cells with LF. Multiple studies have been performed demonstrating that significant anti-tumor activity can be achieved with this intercomplementing toxin in a variety of syngraft, xenograft and orthotopic cancer models. The intercomplementing PrAg consists of PrAg-U2-R200A, a uPA-activated PrAg variant with LF-binding subsite II residue Arg200 mutated to Ala, and PrAg-L1-I210A, a MMP-activated PrAg variant with subsite III residue Ile210 mutated to Ala. PrAg-U2-R200A and PrAg-L1-I210A display reduced cytotoxicity when used singly in combination with LF. However, when combined, they form LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieve high specificity in targeting cells expressing both uPA and MMP activity. Nevertheless, each of these proteins, in particular PrAg-L1-I210A, retains some residual LF-binding ability that lowers the therapeutic index. In collaboration with Steve Leppla, NIAID, we therefore screened a library containing all possible amino acid substitutions for the LF-binding sites on PrAg to find variants with activity strictly dependent upon intermolecular complementation. PrAg-I207R was identified as an excellent replacement for the original variant, PrAg-I210A. Consequently, the new combination of PrAg-L1-I207R and PrAg-U2-R200A retained potent anti-tumor activity, while lowering toxicity to animals, thereby displaying improved therapeutic index. These findings warrant exploration of this novel PrAg-L1-I207R and PrAg-U2-R200A intercomplementing toxin variant as the lead agent for further preclinical and clinical development.